RU2730034C2 - Method of obtaining a stem cell clone suitable for inducing differentiation into somatic cells - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: invention relates to a method of producing secondary stem cell clones from megakaryocyte precursor cells, providing dedifferentiation of precursors of megakaryocytes for formation of a colony of stem cells, as well as separation of secondary stem cells from it. Also disclosed is a method of producing megakaryocyte precursor cells, providing induction of differentiation of recovered clones of secondary stem cells into precursors of megakaryocytes. Invention also relates to a method for producing thrombocytes, providing maturing of the megakaryocyte precursor cells maturation in the megakaryocytes and thrombocyte release.

EFFECT: invention enables efficient production of thrombocytes from megakaryocyte progenitor cells.

10 cl, 11 dwg, 1 tbl, 3 ex

Description

Technology area

[0001] The present invention relates, for example, to a method for obtaining a cell population derived from a single cell, namely cloning, stem cells or somatic cells consisting of different populations, by using reprogramming of somatic cells.

Prior art

[0002] Cloning is an important step in the development of cell lines, and this cloning is routinely performed using the method of serial dilution.

[0003] Although cells are converted to the desired cell type by introducing an exogenous gene into cells (Patent Document 1, Non-Patent Documents 1 and 2), the resulting cells are not uniform due to factors such as differences in the copy number of the introduced exogenous gene or differences in the introduced site in the chromosome. Thus, although it is believed that cloning can be performed according to the limiting dilution method, this limiting dilution method may not necessarily be applied to all cells.

[0004] In addition, since cells obtained by introducing an exogenous gene are less likely to provide the ability to obtain the desired cells again, even if attempts are made to obtain cells in the same way, in the case where the cells require gene manipulation such as homologous recombination , there are limitations of the cell types that allow this manipulation.

[0005] Although replacement therapy has been proposed that involves the induced conversion to T lymphocytes from iPS cells by reprogramming T lymphocytes retaining the desired TCR type (Patent Document 2 or Non-Patent Document 3), this therapy is not performed for cloning or manipulation purposes. genes.

List of links

Patent documents

[0006] Patent Document 1: WO 2014/148646

Patent Document 2: WO 2011/096482

Non-patent documents

[0007] Non-Patent Document 1: Nakamura S, et al, Cell Stem Cell. 14: 535-548, 2014

Non-Patent Document 2: Tanaka A, et al, PLoS One. 8: e61540, 2013

Non-Patent Document 3: Nishimura T, et al., Cell Stem Cell. 12 (1): 114-126, 2013

The essence of the invention

Technical problem

[0008] The object of the present invention is to obtain a cell population derived from a single cell, namely cloning, stem cells or somatic cells composed of different populations.

Solution to the problem

[0009] When the inventors of the present invention isolated stem cells having a gene associated with inducing differentiation into somatic cells included in their chromosome from a colony of stem cells obtained by one or more rounds of dedifferentiation, it was found that the isolated stem cells are suitable for inducing differentiation into somatic cells, thus leading to the implementation of the present invention.

[0010] Namely, the present invention relates to the inventions listed below.

[A1] A method for obtaining a clone of stem cells, which contains the steps:

(i) introducing into stem cells an exogenous gene associated with the induction of differentiation into somatic cells;

(ii) induction of differentiation of stem cells into which the exogenous gene is introduced into somatic cells;

(iii) dedifferentiation of differentiation-induced somatic cells; and

(iv) isolating stem cells carrying an exogenous gene introduced into their chromosome from the stem cell colony formed in step (iii).

[A2] The method described in [A1], where the efficiency of inducing differentiation of isolated clones of stem cells into somatic cells is higher compared to the induction of stem cells before cloning.

[A3] The method described in [A1] or [A2], wherein the somatic cells are hematopoietic progenitor cells, megakaryocytic progenitor cells, erythroblasts, nerve cells, neural stem cells, neural crest cells, myocardial cells, skeletal muscle cells, chondrocytes , hepatocytes or melanocytes.

[A4] The method described in [A3], wherein the somatic cells are precursor cells of megakaryocytes, and the exogenous gene associated with differentiation induction is at least one exogenous gene selected from the group consisting of oncogenes, including genes of the family MYC, genes (polycomb genes) that inhibit the expression of the p16 or p19 gene, including Bmi1, and genes that inhibit apoptosis, including the BCL-XL gene.

[A5] The method described in any one of [A1] to [A4], wherein stem cells expressing MEG3 are isolated.

[A6] The method described in any one of [A1] to [A5], wherein an exogenous gene associated with differentiation induction is operably linked to a drug responsive promoter.

[A7] The method described in any one of items [A1] to [A6], wherein the dedifferentiation in step (iii) is performed by introducing a reprogramming factor selected from the group consisting of OCT3 / 4, SOX2 and KLF4.

[A8] A method for obtaining somatic cells, which comprises the step:

inducing differentiation of stem cell clones obtained in accordance with the method described in any one of items [A1] to [A7] into somatic cells.

[A9] A method for obtaining platelets, which contains the steps:

inducing differentiation of stem cell clones obtained according to the method described in any one of items [A1] to [A6] into megakaryocyte progenitor cells; and

ensuring the maturation of the differentiation-induced megakaryocyte progenitor cells into megakaryocytes and the release of platelets.

[A10] The method described in [A9], wherein the obtained platelets lack HLA.

[0011] [B1] A method for cloning somatic cells, which comprises the steps outlined below, where stem cells are obtained by expression of an exogenous gene:

(i) the formation of a stem cell colony by introducing a reprogramming factor into somatic cells carrying an exogenous gene operably linked to a drug responsive promoter inserted into their chromosome;

(ii) isolating the stem cell colony obtained in step (i); and

(iii) inducing stem cells contained in the stem cell colony isolated in step (ii) to differentiate into somatic cells by contacting cells at any stage of differentiation from stem cells into somatic cells with an appropriate drug.

[B2] The method described in [B1], wherein the somatic cells are precursor cells of megakaryocytes and the exogenous gene is at least one selected from the group consisting of MYC family genes, polycombus genes, and apoptosis suppressing genes.

[B3] The method described in [B2], wherein step (iii) comprises the steps:

(a) inducing stem cells contained in the stem cell colony isolated in step (ii) to differentiate into hematopoietic progenitor cells; and

(b) contacting the hematopoietic progenitor cells obtained in step (a) with an appropriate preparation.

[B4] The method described in any one of [B1] to [B3], wherein the reprogramming factor comprises OCT3 / 4, SOX2, and KLF4.

[B5] The method described in any one of [B1] to [B4], wherein the drug responsive promoter is a promoter having a TRE sequence and further expresses a reverse tetR fusion protein at least in the cells of step (iii).

[B6] The method described in any one of items [B2] to [B5], wherein step (iii) further comprises the step of selecting stem cells expressing MEG3 from among the stem cells contained in the stem cell colony isolated in step (ii) ...

[B7] The method described in any one of items [B1] to [B6], wherein step (iii) further comprises the step of causing the stem cells contained in the stem cell colony isolated in step (ii), there is no HLA.

[B8] The method described in [B7], wherein the HLA is a class I antigen.

[B9] The method described in [B8], wherein the class I antigen is β2-microglobulin.

[B10] A method for obtaining platelets, which contains the steps:

Cloning megakaryocyte progenitor cells using the method described in any one of paragraphs [B2] to [B9]; and

Ensuring the maturation of cloned megakaryocyte progenitor cells into megakaryocytes and the release of platelets.

[B11] A method for obtaining HLA-deficient somatic cells, which comprises the steps:

(i) the formation of pluripotent stem cells by introducing a reprogramming factor into somatic cells;

(ii) initiating that the pluripotent stem cells obtained in step (i) are HLA deficient; and

(iii) inducing the differentiation of HLA-deficient pluripotent stem cells obtained in step (ii) into somatic cells.

[B12] The method described in [B11], wherein the reprogramming factor comprises OCT3 / 4, SOX2, and KLF4.

[B13] The method described in [B11] or [B12], wherein the somatic cells used in step (i) are megakaryocyte progenitor cells, and megakaryocyte progenitor cells are megakaryocyte progenitor cells obtained by incorporating at least , one gene that is functionally linked to a drug-responsive promoter and is selected from the group consisting of genes of the MYC family, polycombus genes, and apoptosis suppressor genes on their chromosome.

[B14] The method described in [B13], wherein step (iii) comprises the steps:

(A) inducing the HLA-deficient pluripotent stem cells obtained in step (ii) to differentiate into hematopoietic progenitor cells; and

(B) contacting the hematopoietic progenitor cells obtained in step (A) with an appropriate preparation.

[B15] The method described in any one of [B11] to [B14], wherein the HLA is a class I antigen.

[B16] The method described in [B15], wherein the class I antigen is β2-microglobulin.

[B17] A method for producing HLA-deficient platelets, which comprises the steps:

obtaining HLA-deficient megakaryocyte progenitor cells using the method described in any one of [B13] to [B16], and

ensuring the maturation of HLA-deficient precursor cells of megakaryocytes into megakaryocytes and the release of platelets.

[B18] iPS cells containing an exogenous oncogene and an exogenous gene that suppresses the expression of the p16 gene or the p19 gene, where the ratio of the content of the exogenous gene that suppresses the expression of the p16 gene or p19 gene and the exogenous oncogene is 2-fold to 7- multiple.

[B19] The iPS cell described in [B18], wherein the oncogene is c-Myc and the gene that suppresses the expression of the p16 gene or the p19 gene is Bmi1.

[B20] A megakaryocyte precursor cell containing an exogenous oncogene and an exogenous gene that suppresses the expression of the p16 gene or the p19 gene, where the ratio of the content of the exogenous gene that suppresses the expression of the p16 gene or p19 gene and the exogenous oncogene is 2-fold to 7- multiple.

[B21] The megakaryocyte progenitor cell described in [B20], where the oncogene is c-Myc and the gene that suppresses the expression of the p16 gene or the p19 gene is Bmi1.

[B22] A method for selecting pluripotent stem cells or hematopoietic progenitor cells suitable for inducing differentiation of megakaryocyte progenitor cells, which comprises the step of: selecting pluripotent stem cells or hematopoietic progenitor cells that express MEG3.

Beneficial effects of the invention

[0012] According to the present invention, a stem cell clone can be obtained that is useful for inducing differentiation into somatic cells. In addition, the stem cells cloned according to the present invention have an excellent proliferative potential compared to stem cells cloned according to the conventional limiting dilution method. For example, not only the secondary clones of megakaryocyte progenitor cells obtained in accordance with the present invention have superior cell proliferation potential and the ability to mature into megakaryocytes compared to conventional clones, megakaryocytes obtained from these clones have a high platelet-forming ability.

Brief Description of Figures

[0013] FIG. 1 is a graph showing the number of platelets produced from 22 candidate megakaryocyte progenitor cell clones obtained by the limiting dilution method along with megakaryocyte progenitor cells (H +) composed of different parent populations (FIG.1A), and the fluorescence intensity instead of the amount of GPIIb / IIIa complex formed by PMA stimulation of platelets (FIG. 1B).

FIG. 2 is a graph showing the results of re-measuring the number of formed platelets obtained from five of the 22 candidate megakaryocyte progenitor cell clones in FIG. 1 along with megakaryocyte progenitor cells (H) composed of different parent populations (FIG. 2A), and fluorescence intensity instead of the amount of GPIIb / IIIa complex formed by PMA stimulation of platelets (FIG. 2B).

FIG. 3 is a graph showing the results of re-measuring the number of formed platelets obtained from 5 clones of megakaryocyte progenitor cells in FIG. 2, along with the original megakaryocyte progenitor cells (H) (Fig. 3A), and the fluorescence intensity instead of the amount of GPIIb / IIIa complex formed by PMA stimulation of platelets (Fig. 3B).

FIG. 4A is a schematic diagram of the production of megakaryocyte progenitor cells, cloning by reprogramming factors, and the production of secondary megakaryocyte progenitor cells from secondary iPS cells. FIG. 4B is a graph showing the copy number per c-MYC or BMI1 cell contained in the chromosomes of secondary iPS cell clones obtained by reprogramming megakaryocyte progenitor cells.

FIG. 5 shows the results of using a flow cytometer to measure the distribution of cells expressing CD42b and CD41a and the distribution of cells expressing CD235 and CD41a among the megakaryocyte progenitor cells obtained from each secondary clone of iPS cells.

FIG. 6A is a schematic diagram of homologous recombination for removal of Exon1 β2-microglobulin. FIG. 6B depicts PCR results to confirm homologous recombination in secondary clones of iPS cells following injection of the target vector.

FIG. 7A shows the results of using a flow cytometer to measure the distribution of β2-microglobulin and HLA expressing cells in megakaryocyte progenitor cells (imMKCL) derived from secondary clones of iPS cells with β2-microglobulin Exon1 removed (shown left), and platelets derived from cells megakaryocyte precursors (shown on the right). FIG. 7B shows the relationship of PAC1 fluorescence intensity after PMA stimulation in platelets derived from megakaryocytes obtained from secondary clones of iPS cells (HLA (-)) with exon1 β2-microglobulin removed or secondary clones of iPS cells (HLA (+)). Fluorescence intensity is presented based on a value of 1 for platelets obtained from secondary clones of iPS cells (HLA (+)).

FIG. 8 shows the results of a comparison of gene expression levels using a microchip commercially available from Illumina (Fig.8A) or Affymetrix (Fig.8B) for secondary clones of iPS cells (good iPS) with the ability to be induced to differentiate into megakaryocyte progenitor cells , and secondary clones of iPS cells (bad iPS), which do not have the ability to be induced to differentiate into megakaryocyte progenitor cells, or hematopoietic progenitor cells obtained from secondary clones of iPS cells (good HPC), which have the ability to be induced to differentiate into cells - precursors of megakaryocytes, and secondary clones of iPS cells (bad HPCs) unable to be induced to differentiate into precursor cells of megakaryocytes.

FIG. 9A and 9B, respectively, shows the growth curves of megakaryocytes (clone 2) obtained from the secondary clone of iPS cells and megakaryocyte progenitors (parental) cells before cloning, and the doubling time calculated from the growth curves (**: P <0.01) ...

FIG. 10A and 10B, respectively, show the appearance of cells and changes in cell size before and after maturation of megakaryocytes (clone 2) derived from the secondary clone of iPS cells and megakaryocyte progenitor cells (parental) before cloning.

FIG. 11 is a graph comparing the number of prothrombocytes, which are progenitors of formed platelets.

Description of embodiments

[0014] The method for obtaining a clone of stem cells according to the present invention comprises the steps:

(i) introducing into stem cells an exogenous gene associated with the induction of differentiation into somatic cells;

(ii) induction of differentiation of stem cells into which the exogenous gene is introduced into somatic cells;

(iii) dedifferentiation of differentiation-induced somatic cells; and

(iv) isolation of stem cells carrying an exogenous gene inserted into their chromosome from the stem cell colony formed in step (iii).

[0015] Isolated clones of stem cells are more suitable to be induced to differentiate into somatic cells compared to stem cells before cloning, and have a high efficiency to be induced to differentiate into somatic cells, on a cell basis.

[0016] In one embodiment, a method for producing a stem cell clone of the present invention may include the steps of:

(i) the formation of a stem cell colony by introducing a reprogramming factor into somatic cells carrying an exogenous gene operably linked to a drug responsive promoter inserted into their chromosome; and

(ii) isolating the stem cell colony obtained in step (i).

[0017] The resulting clone of stem cells can be further induced to differentiate into a somatic cell. Differentiation induction can be performed by a person skilled in the art by a suitable selection of a method suitable for inducing differentiation into a desired somatic cell, and it may not be limited to a particular method, and may further include the step of:

(iii) inducing stem cells contained in the stem cell colony isolated in step (ii) to differentiate into somatic cells by contacting cells at any stage of differentiation from stem cells into somatic cells using an appropriate preparation.

[0018] In the present invention, cloning refers to the cloning of a cell population in the sense of isolating a cell population having uniform genetic information from a cell population having non-uniform genetic information.

[0019] Somatic cells submitted for cloning

In the present invention, there are no particular restrictions on somatic cells submitted for cloning (referred to as primary somatic cells), in cases where the cells are obtained by inserting a gene operably linked to a drug responsive promoter into their chromosome, and examples thereof include nerve cells. cells (WO 2014/148646, Wapinski OL et al, Cell. 155: 621-635, 2013), neural stem cells (Han DW et al, Cell Stem Cell. 10: 465-472, 2012), neural crest cells (Kim YJ, et al, Cell Stem Cell 15: 497-506, 2014), myocardial cells (Ieda M et al, Cell 142: 375-386, 2010), skeletal muscle cells (Tanaka A, et al, PLoS One. 8: e61540, 2013), chondrocytes (Outani H, et al, PLoS One. 8: e77365, 2013), hepatocytes (Huang P, et al, Cell Stem Cell. 14: 370-384, 2014), melanocytes (Yang R , et al, Nat Commun. 5: 5807, 2014), hematopoietic progenitor cells (Batta K, Cell Rep. 9: 1871-84, 2014), erythroblasts (Hirose S, et al, Stem Cell Reports. 1:49 9-508, 2013) and megakaryocyte progenitor cells (Nakamura S, et al, Cell Stem Cell. 14: 535-548, 2014).

[0020] In the present invention, megakaryocyte progenitor cells are useful as somatic cells cloned according to the method of the present invention because they cannot be cloned by the limiting dilution method. Erythroblasts are also useful as somatic cells in the present invention. However, since somatic cells other than these cells also allow the production of stem cell clones carrying an exogenous gene associated with the induction of differentiation into desired somatic cells introduced into their chromosome, somatic cells are not limited to megakaryocyte progenitor cells and erythroblasts.

[0021] An exogenous gene associated with the induction of differentiation into somatic cells in the present invention refers broadly to a gene introduced into a cell to induce differentiation from a stem cell into a somatic cell. To explain this, a gene, when used as an example of the case of somatic cells that are precursor cells of megakaryocytes, the gene associated with differentiation induction may be at least one gene selected from the group consisting of oncogenes, preferably a member of the MYC gene family and more preferably c-Myc, genes that suppress the expression of the p16 gene or the p19 gene (polycomb genes), and preferably Bmi1, and genes that suppress apoptosis, and preferably the BCL-XL gene. Using as an example the case of somatic cells that are erythroblasts, the gene associated with the induction of differentiation can be at least one gene selected from the group consisting of oncogenes, preferably a member of the MYC gene family and more preferably c-Myc, and genes that suppress apoptosis, and preferably the BCL-XL gene. An exogenous gene associated with the induction of differentiation into somatic cells may be operably linked to a drug responsive promoter.

[0022] In the present invention, a drug responsive promoter refers to a promoter that expresses a gene in the presence or absence of a drug. An example of a promoter that expresses a gene in the presence of an appropriate drug is the TRE promoter (CMV minimal promoter carrying the Tet response sequence, including seven repeats of the tet0 sequence). In the case of using the TRE promoter, a system is preferably used in which gene expression is induced in the presence of an appropriate drug (such as tetracycline or doxycycline) by a concurrently expressed fusion protein (reverse tetR fusion protein) of reverse tetR (rtetR) and VP16AD in the same cells. In the case of using a reverse tetR fusion protein, it is necessary that the fusion protein is at least expressed in the "step of inducing differentiation from stem cells into secondary somatic cells", which will be described later. For example, through a functionally binding gene encoding a reverse tetR fusion protein to a drug responsive promoter and introducing this gene upon receipt of primary somatic cells, the reverse tetR fusion protein can be expressed by adding or removing an appropriate drug during the "step of inducing differentiation from stem cells into secondary somatic cells. cells ". In the case of an operably linked drug responsive promoter with two or more types of genes, the same type of drug responsive promoter can be used for all genes, or two or more types of drug responsive promoters can be used.

[0023] A method of obtaining primary progenitor cells of megakaryocytes

[0024] In the present invention, "megakaryocyte progenitor cells" refer to cells that become megakaryocytes as a result of maturation. These cells are not multinucleated, and include cells characterized as CD41a positive / CD42a positive / CD42b weakly positive. The megakaryocyte progenitor cells of the present invention are preferably cells that can be grown by expansive cultivation, such as cells capable of undergoing expansive cultivation for at least 60 days under suitable conditions. In the present invention, megakaryocyte progenitor cells may or may not be cloned, and although there are no particular restrictions on this, those that have been cloned are referred to as megakaryocyte progenitor cell line. The megakaryocyte progenitor cells in the present invention can be obtained from hematopoietic progenitor cells.

[0025] In the present invention, "megakaryocytes", which are also referred to as progenitor cells and megakaryocytic cells, are cells that produce platelets by separation from their cytoplasm, may be multi-nucleus cells, and include cells characterized as, for example, CD41a- positive / CD42a-positive / CD42b-positive. In addition, megakaryocytes can also be characterized as expressing GATA1, FOG1, NF-E2 and β1-tubulin. Multinucleated megakaryocytes refer to cells or a group of cells in which the number of nuclei has undergone a relative increase in comparison with the precursor cells of megakaryocytes. For example, in the case where the nuclei of the megakaryocyte progenitor cells to which the method of the present invention is applied are 2N, the cells in which the nuclei are 4N or more are multinucleated megakaryocytes. In addition, in the present invention, megakaryocytes can be immortalized as a megakaryocytic cell line, or can be a cloned group of cells.

[0026] In the present invention, hematopoietic progenitor cells (HPC) refer to cells capable of differentiating into blood cells such as lymphocytes, eosinophils, neutrophils, basophils, erythrocytes, or megakaryocytes, and in the present invention, there is no separation between hematopoietic progenitor cells and hematopoietic stem cells, and this refers to the same cells, unless otherwise indicated. Hematopoietic stem / progenitor cells can be recognized by, for example, a positive trait for surface antigens, CD34 and / or CD43. In the present invention, hematopoietic stem cells can also be applied to hematopoietic progenitor cells that have been induced to differentiate from pluripotent stem cells or hematopoietic stem cells, as well as progenitor cells derived from placental blood, bone marrow blood, or peripheral blood. For example, in the case of using pluripotent stem cells, hematopoietic progenitor cells can be obtained from a net-like structure (ES bag or iPS bag) obtained by culturing pluripotent stem cells on C3H10T1 / 2 in the presence of VEGF according to the method described in Takayama N ., et al. J Exp Med. 2817-2830 (2010). Here, a "net-like structure" refers to a three-dimensional bag-like structure (having a space inside) obtained from pluripotent stem cells, which is formed by a population of endothelial cells or the like and contains hematopoietic progenitor cells in its interior. Other examples of methods used to induce differentiation from pluripotent stem cells into hematopoietic progenitor cells include a method that uses embryoid body formation and cytokine addition (Chadwick et al. Blood 2003, 102: 906-15, Vijayaragavan et al. Cell Stem Cell 2009, 4: 248-62, Saeki et al. Stem Cells 2009, 27: 59-67), and a method involving co-cultivation with stromal cells derived from various species (Niwa et al. J Cell Physiol. 2009 Nov ; 221 (2): 367-77.).

[0027] Examples of pluripotent stem cells include fertilized eggs and cells such as embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), or embryonic germ cells (EG cells). The precursor cells of megakaryocytes serving as somatic cells of the present invention are preferably those that have been induced by a cell culture step by overexpression of an oncogene, a gene that suppresses the expression of the p16 gene or the p19 gene (polycomb gene) and / or a gene that suppresses apoptosis in hematopoietic progenitor cells.

[0028] In the present invention, "oncogene" refers to a gene that causes malignant transformation of normal cells as a result of expression, their structure or function, which is different from the structure and function of normal cells, and examples thereof include genes of the MYC family, genes of the Src family, genes of the Ras family, genes of the Raf family, genes of the c-Kit family and protein kinases such as PDGFR or Abl. Examples of genes in the MYC family include c-MYC, N-MYC, and L-MYC. The c-MYC genes refer, for example, to genes consisting of a nucleic acid sequence as provided in the NCBI, accession number. NM 002467. In addition, c-MYC genes include their homologues, and homologues of c-MYC genes refer to genes for which, for example, their cDNA sequence consists of a sequence that is substantially identical to the nucleic acid sequence presented in NCBI, number Accession NM 002467. cDNA consisting of a sequence substantially identical to the nucleic acid sequence provided by the NCBI, Accession NM 002467 refers to DNA consisting of a sequence having an identification of about 60% or more, preferably about 70% or more, more preferably about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%, and most preferably about 99%, with DNA consisting of the sequence shown in NCBI accession number NM 002467, or DNA capable of hybridizing under stringent conditions to DNA consisting of a sequence complementary to the sequence the nucleic acid provided by the NCBI, accession number NM 002467, with the protein encoded by these DNAs, facilitating the expansion of cells such as hematopoietic progenitor cells that are in the stage of differentiation.

[0029] Here, stringent conditions refer to hybridization conditions readily determined by one of ordinary skill in the art, which are generally empirical experimental conditions, depending on the length of the probe, the washing temperature, and the salt concentration. In general, the temperature for proper annealing gets higher with increasing probe length, and the temperature gets lower with decreasing probe length. The formation of a hybrid, as a rule, depends on the ability of the complementary strand to undergo repeated annealing in the external environment at a temperature slightly lower than their melting point.

[0030] For example, an example of a slightly stringent condition includes washing at 0.1 x SSC in 0.1% SDS solution at a temperature condition of 37 ° C to 42 ° C during the post-hybridization filter wash step. In addition, an example of a high severity condition includes a 5 x SSC wash in 0.1% SDS solution at 65 ° C during the wash step. Polynucleotides of higher homology can be obtained by using more stringent conditions.

[0031] In the present invention, since it is preferable to suppress the expression level of c-MYC, c-MYC may be one that encodes a protein fused to a destabilizing domain. A destabilizing domain purchased from ProteoTuner or Clontech Laboratories, Inc. can be used.

[0032] In the present invention, examples of "genes that suppress the expression of the p16 gene or the p19 gene" include BMI1, Id1, Mel18, Ring1a / b, Phc1 / 2/3, Cbx2 / 4/6/7/8, Ezh2, Eed, Suz12, HDAC and Dnmt1 / 3a / 3b. The BMI1 gene refers, for example, to a gene consisting of the nucleic acid sequence provided in NCBI, accession number NM 005180. In addition, the BMI1 gene includes homologues thereof, and BMI1 gene homologues refer to genes for which their cDNA sequence consists of the sequence substantially identical to the nucleic acid sequence provided by the NCBI, accession number NM 005180. cDNA consisting of a sequence substantially identical to the nucleic acid sequence provided by the NCBI, accession number NM 005180 refers to DNA consisting of a sequence having an identification of approximately 60% or more, preferably about 70% or more, more preferably about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97% or 98%, and most preferably about 99%, with DNA consisting of the sequence shown in NCBI accession number NM 005180, or DNA capable of hybridizing under stringent conditions with DNA consisting of a sequence complementary to the nucleic acid sequence presented in NCBI, accession number NM 005180, with a protein encoded by DNA that stimulates cell expansion by suppressing cell senescence, capable of inducing oncogenes present in cells that are expressed oncogenes such as the MYC family genes.

[0033] In the present invention, “genes that suppress apoptosis” refer to genes that suppress apoptosis, there is no limitation thereto, and examples thereof include the BCL2 gene, BCL-XL gene, survivin, and MCL1. The BCL-XL gene refers to a gene consisting of the nucleic acid sequence shown in NCBI accession number NM 001191 or NM 138578. In addition, the BCL-XL gene includes homologues thereof, and BCL-XL gene homologues refer to genes for which, for example, their cDNA sequence consists of a sequence that is substantially identical to the nucleic acid sequence provided in NCBI accession number NM 001191 or NM 138578. cDNA consisting of a sequence substantially identical to the nucleic acid sequence provided in NCBI accession number NM 001191 or NM 138578 refers to DNA consisting of a sequence having an identification of about 60% or more, preferably about 70% or more, more preferably about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98%, and most preferably about 99%, with DNA consisting of the sequence, represented in the NCBI, accession number NM 001191 or NM 138578, or DNA capable of hybridizing under stringent conditions to DNA consisting of a sequence complementary to the nucleic acid sequence provided by the NCBI, accession number NM 001191 or NM 138578, with a protein encoded by this DNA having an inhibitory effect apoptosis.

[0034] In the present invention, the method for overexpressing the above genes in hematopoietic progenitor cells is preferably implemented by inserting a gene operably linked to a drug responsive promoter on its chromosome, and can be achieved by, for example, introducing an expression vector containing the gene, functionally linked to a drug responsive promoter into a hematopoietic progenitor cell. Examples of vectors expressing these genes that can be used include retrovirus, lentivirus, and other viral vectors, as well as animal cell expression plasmids (such as pA1-11, pXT1, pRc / cmV, pRc / RSV, or pcDNAI / Neo). Retroviral vectors or lentiviral vectors are preferably used from the point of view of chromosome integration.

[0035] In addition to a promoter, the expression vector may contain an enhancer, an additional poly (A) signal, a selectable marker gene or SV40 origin of replication, and the like. Examples of suitable selectable marker genes include a dihydrofolate reductase gene, a neomycin resistance gene, and a puromycin resistance gene.

[0036] In the present invention, a polycistronic vector in which genes are linked longitudinally can be prepared to introduce multiple genes at the same time. To provide polycistronic expression, the sequence 2A of the self-cleaving peptide of the leg and mouth virus (reference, for example, Science, 322, 949-953, 2008), or IRES (internal ribosome binding site), can be sutured between many overexpressed genes.

[0037] In the present invention, in the case of a viral vector, a method of introducing an expression vector into hematopoietic progenitor cells can be performed by introducing a plasmid containing a nucleic acid into suitable packaging cells (such as Plat-E cells) or a complementary cell line (such as cells 293), followed by recovery of the virus produced in the cell culture supernatant, and infection of hematopoietic progenitor cells by contact with the virus. In the case of a non-viral vector, the plasmid vector can be introduced into cells using a method such as lipofection, liposomal method, electroporation, calcium phosphate co-precipitation, DEAE-dextran method, microinjection, or gene gun.

[0038] In one aspect of the method for inducing megakaryocyte progenitor cells of the present invention, a gene that suppresses apoptosis may be overexpressed after overexpressing an oncogene or a gene that suppresses expression of the p16 gene or p19 gene in hematopoietic progenitor cells. Overexpression of a gene that suppresses apoptosis can be carried out in the same manner as described above by introducing an expression vector, a protein encoded by these genes, or an RNA encoding these genes into hematopoietic progenitor cells. In the case of subsequent expression of the apoptosis suppressor gene, although there is no limitation thereto, overexpression of the apoptosis suppressor gene is preferably performed after overexpression of the oncogene or the p16 gene or p19 gene suppressive gene for at least 14 days.

[0039] In the present invention, a caspase inhibitor can be contacted with hematopoietic progenitor cells in place of the over-expressing apoptosis suppressing gene in the cells. In the present invention, the caspase inhibitor can be any peptide compound, non-peptide compound, or biological protein. Examples of peptide compounds include the artificial chemically synthesized peptide compounds specified in items (1) to (10) below.

(1) Z-Asp-CH2-DCB (molecular weight: 454.26)

(2) Boc-Asp (OMe) -FMK (molecular weight: 263.3)

(3) Boc-Asp (OBzl) -CMK (molecular weight: 355.8)

(4) Ac-AAVALLPAVLLALLAP-YVAD-CHO (molecular weight: 1990.5) (SEQ ID NO: 1)

(5) Ac-AAVALLPAVLLALLAP-DEVD-CHO (molecular weight: 2000.4) (SEQ ID NO: 2)

(6) Ac-AAVALLPAVLLALLAP-LEVD-CHO (molecular weight: 1998.5) (SEQ ID NO: 3)

(7) Ac-AAVALLPAVLLALLAP-IETD-CHO (molecular weight: 2000.5) (SEQ ID NO: 4)

(8) Ac-AAVALLPAVLLALLAP-LEHD-CHO (molecular weight: 2036.5) (SEQ ID NO: 5)

(9) Z-DEVD-FMK (Z-Asp-Glu-Val-Asp-fluoromethyl ketone) (SEQ ID NO: 6)

(10) Z-VAD FMK

[0040] Examples of peptide compound caspase inhibitors include: (1) VX-740 - Vertex Pharmaceuticals (Leung-Toung et al., Curr. Med. Chem. 9, 979-1002 (2002)) and (2) HMR-3480 - Aventis Pharma AG (Randle et al., Expert Opin. Investig. Drugs 10, 1207-1209 (2001)).

[0041] Examples of non-peptide compound caspase inhibitors include: (1) anilinoquinazolines (AQZs), AstraZeneca Pharmaceuticals (Scott et al., J. Pharmacol. Exp. Ther. 304, 433-440 (2003)), (2) M826 - Merck Frosst (Han et al., J. Biol. Chem. 277, 30128-30136 (2002)), (3) M867 - Merck Frosst (Methot et al., J. Exp. Med. 199, 199-207 (2004) ), and (4) nicotinyl aspartyl ketones - Merck Frosst (Isabel et al., Bioorg. Med. Chem. Lett. 13, 2137-2140 (2003)).

[0042] In addition, examples of caspase inhibitors of other non-peptide compounds include: (1) IDN-6556 - Idun Pharmaceuticals (Hoglen et al., J. Pharmacol. Exp. Ther. 309, 634-640 (2004)), (2) MF-286 and MF-867 - Merck Frosst (Los et al., Drug Discov. Today 8, 67-77 (2003)), (3) IDN-5370 - Idun Pharmaceuticals (Deckwerth et al., Formulation Dev. Res. 52, 579-586 (2001)), (4) IDN-1965 - Idun Pharmaceuticals (Hoglen et al., J. Pharmacol. Exp. Ther. 297, 811-818 (2001)), and (5) VX-799 - Vertex Pharmaceuticals (Los et al., Drug Discov. Today 8, 67-77 (2003)). Other examples of caspase inhibitors include M-920 and M-791 - Merck Frosst (Hotchkiss et al., Nat. Immunol. 1, 496-501 (2000)).

[0043] In the present invention, the caspase inhibitor is preferably Z-VAD FMK. In the case of using the Z-VAD FMK for a caspase inhibitor, the Z-VAD FMK is added to the medium in which the hematopoietic progenitor cells are cultured. The preferred concentration of Z-VAD FMK in the medium is, for example, 10 µM or more, 20 µM or more, 30 µM or more, 40 µM or more or 50 µM or more, and is preferably 30 µM or more.

[0044] Although not limited in this respect, the medium used to obtain megakaryocyte progenitor cells from hematopoietic progenitor cells can be obtained by using the medium used for culturing animal cells as the minimal medium. Examples of minimal media include IMDM medium, 199 medium, Eagle's minimal maintenance medium (EMEM), αMEM medium, Dulbecco's modified Eagle's medium (DMEM), Ham's F12 medium, RPMI 1640 medium, Fischer's medium, neurominimal medium (Life Technologies) and their mixed medium. ... The medium may contain serum or may be serum free. The medium may also contain one or more substances such as albumin, insulin, transferrin, selenium, fatty acids, trace elements, 2-mercaptoethanol, thiol glycerin, lipid, amino acids, L-glutamine, non-essential amino acids, vitamins, growth factors, compounds with low molecular weight, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts or cytokines as needed. Cytokines refer to proteins that stimulate hematopoietic differentiation and examples include VEGF, TPO, and SCM. The preferred medium in the present invention is IMDM medium containing serum, insulin, transferrin, serine, thiol glycerol, ascorbic acid, and TPO. The medium more preferably further comprises SCM. In addition, in the case of using an expression vector containing a drug responsive promoter, the corresponding drug such as tetracycline or doxycycline is preferably contained in the medium in an overexpression step.

[0045] In the present invention, although there is no limitation in this regard, the temperature conditions for obtaining megakaryocyte progenitor cells from hematopoietic progenitor cells are such that the stimulation of differentiation into megakaryocyte progenitor cells is maintained by culturing the hematopoietic progenitor cells at a temperature 37 ° C or higher. Here, since a temperature that does not damage cells is suitable, a temperature of 37 ° C or higher refers to, for example, a temperature of about 37 ° C to about 42 ° C, and preferably a temperature of about 37 ° C to about 39 ° C. The duration of cultivation at a temperature of 37 ° C or higher can be suitably determined by the person skilled in the art while monitoring factors such as the number of megakaryocyte progenitor cells. Although there is no particular limitation on this duration, the desired number of megakaryocyte progenitor cells are obtained, examples thereof include a duration of at least 6 days or more, 12 days or more, 18 days or more, 24 days or more, 30 days or more, 42 days or more, 48 days or more, 54 days or more, or 60 days or more, and preferably 60 days or more. The long culture period does not present a problem with respect to the induction of megakaryocyte progenitor cells. Moreover, it is preferable to suitably subculture during the cultivation period.

[0046] A method for reprogramming somatic cells

In the present invention, the introduction of a reprogramming factor into somatic cells can be carried out for the method used for reprogramming somatic cells. Here, examples of reprogramming factors include genes or gene products such as Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas , ECAT15-2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, or Glis1, and these reprogramming factors can be used alone or in combination. Examples of combinations of reprogramming factors include those described in WO 2007/069666, WO 2008/118820, WO 2009/007852, WO 2009/032194, WO 2009/058413, WO 2009/057831, WO 2009/075119, WO 2009/079007, WO 2009/091659, WO 2009/101084, WO 2009/101407, WO 2009/102983, WO 2009/114949, WO 2009/117439, WO 2009/126250, WO 2009/126251, WO 2009/126655, WO 2009/157593, WO 2010/009015, WO 2010/033906, WO 2010/033920, WO 2010/042800, WO 2010/050626, WO 2010/056831, WO 2010/068955, WO 2010/098419, WO 2010/102267, WO 2010/111409, WO 2010/111422, WO 2010/115050, WO 2010/124290, WO 2010/147395, WO 2010/147612, Huangfu D, et al. (2008) Nat. Biotechnol. 26: 795-797, Shi Y, et al. (2008) Cell Stem Cell 2: 525-528, Eminli S, et al. (2008), Stem Cells. 26: 2467-2474, Huangfu D, et al. (2008) Nat. Biotechnol. 26: 1269-1275, Shi Y, et al. (2008) Cell Stem Cell, 3, 568-574, Zhao Y, et al. (2008), Cell Stem Cell, 3: 475-479, Marson A, (2008), Cell Stem Cell, 3, 132-135, Feng B, et al. (2009), Nat. Cell Biol. 11: 197-203, R. L. Judson et al., (2009), Nat. Biotechnol., 27: 459-461, Lyssiotis CA, et al. (2009) Proc Natl Acad Sci USA. 106: 8912-8917, Kim JB, et al. (2009) Nature. 461: 649-643, Ichida JK, et al. (2009) Cell Stem Cell. 5: 491-503, Heng JC, et al. (2010) Cell Stem Cell. 6: 167-74, Han J, et al. (2010) Nature. 463: 1096-100, Mali P, et al. (2010), Stem Cells. 28: 713-720 and Maekawa M, et al. (2011) Nature. 474: 225-9. A more preferred combination of reprogramming factors includes Oct3 / 4, Sox2, and Klf4.

[0047] The above reprogramming factors include factors used to enhance efficacy, such as histone deacetylase (HDAC) inhibitors (such as low molecular weight inhibitors like valproic acid (VPA), trichostatin A, sodium butyrate, MC 1293 or M344, expression inhibitors nucleic acids such as anti-HDAC siRNA and shRNA (e.g. HDAC1 siRNA Smartpool® (Millipore), HuSH 29mer anti-HDAC1 shRNA constructs (Ori gene)), MEK inhibitors (such as PD184352, PD98059, U0126, SL327 or PD0325901 inhibitors) glycogen synthase kinase-3 (such as Bio or CHIR99021), DNA methyl transferase inhibitors (such as 5-azacytidine), histone methyl transferase inhibitors (such as small molecule inhibitors such as BIX-01294 or inhibitors of nucleic acid expression such as siRNA and kshRNA Suv39h2, SetDBl or G9a), calcium L-channel agonists (such as Bayk8644), butyric acid, TGFβ inhibitors or ALK5 inhibitors (such as LY364947, SB431542, 616453 or A-83-01), p53 inhibitors (such as siRNA and anti-p53 shRNA), ARID3A inhibitors (such as siRNA and anti-ARID3A shRNA), miRNAs such as miR-291-3p, miR-294 , miR-295 or miR-302), Wnt signaling (such as soluble Wnt3a), neuropeptide Y, prostaglandins (such as prostaglandin E2 or prostaglandin J2), hTERT, SV40LT, UTF1, IRX6, GLISl, PITX2 or DMRTBl, and in the present description does not make specific distinctions between reprogramming factors, and these factors are used to improve the established efficiency.

[0048] In the case where the reprogramming factor is a protein, the reprogramming factor can be introduced into somatic cells by methods such as lipofection, fusion with a cell penetrating peptide (such as TAT or polyarginine derived from HIV), or microinjection.

[0049] On the other hand, in the case where the reprogramming factor is DNA, DNA can be introduced into somatic cells through a vector like a virus, plasmid, or artificial chromosome, and through methods such as lipofection, liposomes or microinjection. Examples of viral vectors include retroviral vector, lentiviral vector (described in Cell, 126, pp. 663-676, 2006; Cell, 131, pp. 861-872, 2007; Science, 318, pp. 1917-1920, 2007), adenoviral a vector (Science, 322, 945-949, 2008), an adeno-associated viral vector, and a Sendai viral vector (WO 2010/008054). In addition, examples of artificial chromosome vectors include human artificial chromosomes (HAC), yeast artificial chromosomes (YAC), and bacterial artificial chromosomes (BAC, PAC). Mammalian cell plasmids can be used as plasmids (Science, 322: 949-953, 2008). The vectors may contain a control sequence, such as a promoter, enhancer, ribosome binding sequence, terminator, or polyadenylation site to permit expression of the nuclear reprogramming agent, and may further contain a drug resistance gene (such as a kanamycin resistance gene, ampicillin resistance gene, or a gene puromycin resistance), a selectable marker sequence such as a thymidine kinase gene or a diphtheria toxin gene, or a reporter sequence gene such as green fluorescent protein (GFP), β-glucuronidase (GUS) or FLAG, as appropriate. In addition, the above vectors may have a LoxP sequence before or after the vector to remove genes encoding reprogramming factors, or both a promoter and a gene encoding a reprogramming factor associated therewith, after introduction into somatic cells.

[0050] In the case where the reprogramming factor is in the form of an RNA, the reprogramming factor can be introduced into somatic cells by a method such as lipofection or microinjection, and an RNA with embedded 5-methylcytidine and pseudouridine (TriLink Biotechnologies) can be used to inhibit degradation (Warren L, (2010) Cell Stem Cell. 7: 618-630).

[0051] Examples of cell culture fluid after reprogramming include DMEM containing 10% to 15% FBS, DMEM / F12 and DME culture fluid (and these culture fluid may suitably further contain LIF, penicillin / streptomycin, puromycin, L-glutamine , nonessential amino acids or β-mercaptoethanol, etc.), as well as commercially available culture broths (such as mouse ES cell culture broth (TX-WES culture broth, Thrombo-X), primate ES cell culture broth ( ES primate / iPS cell culture broth, ReproCELL Inc.), or serum-free medium (mTeSR, Stemcell Technology)).

[0052] An example of a method for culturing cells after reprogramming comprises contacting somatic cells with a reprogramming factor in DMEM containing 10% FBS or DMEM / F12 culture fluid at 37 ° C in the presence of 5% CO2 and culturing for about 4 days to 7 days , followed by subculture of cells onto feeder cells (such as STO cells or SNL cells treated with mitomycin C) and culturing in a primate ES cell culture broth containing bFGF, starting approximately 10 days after contacting the somatic cells with a reprogramming factor to ensure the formation of iPS-like colonies after about 30 days to about 45 days or more from the contact time.

[0053] Alternatively, somatic cells are cultured in DMEM medium containing 10% FBS (which may also suitably contain LIF, penicillin / streptomycin, puromycin, L-glutamine, nonessential amino acids or β-mercaptoethanol, etc.) on feeder cells (such as STO cells or SNL cells treated with mitomycin C) at 37 ° C in the presence of 5% CO2 to ensure the formation of ES-like colonies after about 25 days to about 30 days. Reprogrammed somatic cells are preferably used as is instead of feeder cells (Takahashi K, et al. (2009), PLoS One. 4: e8067 or WO 2010/137746), or an extracellular matrix (such as laminin-5 (WO 2009/123349) or Matrigel (Becton, Dickinson and Company)).

[0054] In addition, examples of culture methods include methods using a medium that does not contain serum (Sun N, et al. Proc Natl Acad Sci USA. 106: 15720-15725, 2009 or Nakagawa M, et al, Sci Rep. 4 : 3594, 2014). In addition, iPS cells can be housed under hypoxic conditions (oxygen concentration from 0.1% to 15%) to improve containment efficiency (Yoshida Y, et al. (2009), Cell Stem Cell. 5: 237-241 or WO 2010 / 013845).

[0055] The culture fluid is replaced with fresh culture fluid once a day, starting from 2 days after the start of the culture during the above culture. In addition, although there is no particular limitation in this regard, an example of the number of somatic cells used in reprogramming is in the range from about 5 × 10 ³ to about 5 × 10 ⁶ cells per 10 cm2 of the culture dish area.

[0056] The step of isolating stem cell colonies obtained by reprogramming somatic cells

In the present invention, stem cell colonies can be obtained by introducing a reprogramming factor into somatic cells and culturing the cells as described above. In the present invention, stem cells refer to cells that have the ability to self-replicate, which allows cells to form cells identical to those cells through cell division, and the ability to differentiate into different types of cells, while having the ability to proliferate without limitation. Although there are no particular restrictions on the stem cells of the present invention, they form colonies, these stem cells are pluripotent stem cells with the ability to differentiate into tissue cells, excluding placental cells.

[0057] In the present invention, a colony refers to a cell mass obtained from a single cell.

[0058] The cloning method according to the present invention comprises the step of isolating the obtained stem cell colony. This isolation can be carried out by suitably collecting a single colony and then transferring to another culture vessel.

[0059] iPS cells for inducing megakaryocyte progenitor cells

In the present invention, in case the somatic cells are megakaryocyte progenitor cells, an exogenous oncogene and an exogenous gene suppressing p16 gene expression or a p19 gene operably linked to a drug responsive promoter may be contained in the chromosomes of megakaryocyte progenitor cells, as previously described ... In this case, secondary iPS cells obtained by reprogramming megakaryocyte progenitor cells in accordance with the method described above similarly contain an exogenous oncogene and an exogenous gene that suppresses the expression of the p16 gene or the p19 gene, functionally linked to the drug-responsive promoter in their chromosomes. ... At the moment, the ratio of the content of an exogenous gene suppressing the expression of the p16 gene or of the p19 gene operably linked to a drug responsive promoter and an exogenous oncogene operably linked to a drug responsive promoter in iPS cells for inducing megakaryocyte progenitor cells is preferably from 2 -fold to 7-fold, and more preferably from 3-fold to 5-fold. Similarly, the ratio of exogenous gene suppressing expression of p16 gene or p19 gene operably linked to a drug responsive promoter to an exogenous oncogene operably linked to drug responsive promoter in megakaryocyte progenitor cells is preferably 2-fold to 7- multiple, and more preferably from 3 to 5 times. In addition, the oncogene and the gene that suppresses the expression of the p16 gene or the p19 gene are suitably selected from the above genes.

[0060] The step of inducing differentiation from stem cells into secondary somatic cells

The cloning method of the present invention comprises the step of inducing differentiation of stem cells obtained according to the method described above into secondary somatic cells. In the present invention, secondary somatic cells refers to somatic cells obtained by reprogramming primary somatic cells into stem cells, followed by induction to differentiate into secondary somatic cells, and the primary somatic cells and the secondary somatic cells are preferably the same cells. This induction step can be carried out by re-expression of the inserted gene. Gene re-expression can be carried out by contacting cells at any stage of differentiation from stem cells obtained by reprogramming primary somatic cells to secondary somatic cells using an appropriate drug (in the case of a promoter that expresses the gene in the presence of an appropriate drug), or by interrupting contact between cells at any stage of differentiation from stem cells obtained by reprogramming primary somatic cells into secondary somatic cells and an appropriate drug (in the case of a promoter that expresses a gene when the corresponding drug is removed). For example, in the case of a fusion gene (reverse tetR) rtetR and VP16AD, the gene can be re-expressed by administering an appropriate drug. "Cells at any stage of differentiation from stem cells obtained by reprogramming primary somatic cells to secondary somatic cells" can be any cells in which the stem cells have passed through differentiation to secondary somatic cells. Thus, in the present invention, stem cells can be induced to differentiate into other cells prior to gene re-expression, and examples of cells susceptible to this differentiation induction include fibroblasts and hematopoietic progenitor cells. The induction of differentiation into "other cells" can be carried out in accordance with known methods. In the case of using megakaryocyte progenitor cells as somatic cells, the previously described method for inducing differentiation into hematopoietic progenitor cells is an example of a method used for inducing differentiation. Namely, by administering a drug corresponding to the medium used to induce differentiation from stem cells into hematopoietic progenitor cells and induce differentiation from previously described hematopoietic progenitor cells into megakaryocyte progenitor cells, an oncogene, a gene that suppresses the expression of the p16 gene or p19 gene, and / or the apoptosis suppressing gene may be overexpressed.

[0061] The step of selecting stem cells or hematopoietic progenitor cells

In the present invention, since all stem cells are not necessarily capable of being induced to differentiate into megakaryocyte progenitor cells when megakaryocyte progenitor cells are used as somatic cells, it is preferable to appropriately select stem cells capable of being induced to differentiate into megakaryocyte progenitor cells, and an example of a method for carrying out this procedure comprises selecting those stem cells that express MEG3. In the present invention, hematopoietic progenitor cells derived from stem cells expressing MEG3 can also be selected. In the present invention, in the case of humans, MEG3 refers to a non-coding RNA consisting of a nucleic acid sequence as shown in NCBI, accession number NR 002766, NR 003530, NR 003531, NR 033358, NR 033359, NR 033360, NR 046464, NR 046465, NR 046466, NR 046467, NR 046468, NR 046469, NR 046470, NR 046471, NR 046472 or NR 046473. Although there are no particular restrictions in this regard, the stem cells to which the method of the present invention is applied may be primary pluripotent stem cells, and more preferably stem cell clones obtained according to the method described above. A high level of expression may refer to expression at a level that is higher than the mean in a plurality of simultaneously measured stem cells or hematopoietic progenitor cells, or may refer to an expression level that is higher than that of known stem cells or hematopoietic progenitor cells. which cannot be induced to differentiate into megakaryocyte progenitor cells.

[0062] In the present invention, a method known to those skilled in the art can be used as a method for confirming MEG3 expression, and examples thereof include reverse transcription PCR analysis, quantitative reverse transcription PCR analysis, Northern blot analysis, immunohistochemical analysis, analysis on a microchip and their combinations.

[0063] A method of initiating somatic cells to be HLA deficient and a method of producing HLA-deficient somatic cells

In one embodiment, the present invention provides a method for initiating somatic cells to be HLA deficient, which includes the following steps, or a method for producing HLA deficient somatic cells:

(i) the formation of pluripotent stem cells by introducing a reprogramming factor into somatic cells;

(ii) initiation of pluripotent stem cells obtained in step (i) to be HLA deficient; and

(iii) induction of HLA-deficient pluripotent stem cells obtained in step (ii) to differentiate into somatic cells.

[0064] The somatic cells provided for use in the method for inducing HLA deficiency of the present invention, the reprogramming factor used, the method for generating pluripotent stem cells, and the method for inducing pluripotent stem cells to differentiate into somatic cells are the same as in the case of somatic cells, provided for use in the above cloning. Thus, the present invention also relates to a method for cloning somatic cells and further causing those cells to be HLA deficient.

[0065] In the present invention, HLA refers to a human lymphocyte antigen, and refers to a class I antigen consisting of an α chain and an L chain, a class II antigen consisting of a β chain encoded by the DRB1 gene and an α chain encoded by the DRA gene, and class III antigen. Since the expressed HLA differs according to the somatic cell, the HLA to be removed can be suitably selected, and in the case of using megakaryocyte progenitor cells as somatic cells, the HLA is preferably selected and the class I antigen is removed. Removal of HLA refers to the removal of the α chain. , L chain or β chain, and in case of removal of the class I antigen, preferably the L chain, namely β2-microglobulin, is removed.

[0066] The method of initiating a chromosome to be HLA deficient in pluripotent stem cells of the present invention can be carried out by a suitably selected known method such as homologous recombination.

[0067] Method for producing platelets

The method for producing platelets of the present invention comprises the step of cloning megakaryocyte progenitor cells using the cloning method of the present invention, and the step of allowing the cloned megakaryocyte progenitor cells to mature into megakaryocytes and release the platelets. The step of allowing the cloned megakaryocyte progenitor cells to mature and release the platelets can be carried out according to a known method or a method corresponding thereto. For example, in the case where megakaryocyte progenitor cells contain at least one gene selected from the group consisting of the MYC family genes, polycomb genes and genes that suppress apoptosis, the maturation of megakaryocytes can be carried out by suppressing the expression of genes of the MYC family, polycomb genes and / or genes that suppress apoptosis, by removing the corresponding drug from the environment after the above step (iii). Ripe megakaryocytes become multinucleated and release platelets.

[0068] The platelets can be in the form of a platelet preparation by combining with ACD-A solution, FFP, sodium citrate, citric acid or glucose, and the like, or can be in the form of a blood preparation by combining with erythrocytes.

[0069] In the case of obtaining HLA-deficient megakaryocyte clones according to the method described above, HLA-deficient platelets can be obtained by allowing the megakaryocyte clones to mature and release the platelets. HLA-deficient platelets are useful because they can be transfused regardless of the HLA type of recipient.

[0070] A method for improving the proliferative capacity of megakaryocyte progenitor cells

The present invention relates to a method for improving the proliferative capacity of megakaryocyte progenitor cells by obtaining stem cells by reprogramming megakaryocyte progenitor cells and subsequent conversion into megakaryocyte progenitor cells. Thus, in one of its embodiments, the method for improving the proliferative capacity of megakaryocyte progenitor cells of the present invention comprises the steps of:

(i) the formation of a stem cell colony by introducing a reprogramming factor into megakaryocyte progenitor cells carrying an exogenous gene that is expressed in response to the drug;

(ii) isolating the stem cell colony obtained in step (i); and

(iii) inducing stem cells contained in the stem cell colony isolated in step (ii) to differentiate into megakaryocyte progenitor cells, where induction into somatic cells comprises the step of contacting the appropriate drug.

[0071] In the present invention, the improvement in proliferative ability refers to an increase in the length of the telomeric sequence in the chromosome. In the present invention, a telomeric sequence refers to a repeating sequence including TTAGGG, and an increase in telomeric sequence length means that the number of repeats has increased.

Examples of

[0072] Although the following is a more detailed explanation of the present invention based on examples and test examples, the present invention is not limited to the following examples.

[0073] Obtaining megakaryocyte progenitor cells

Hematopoietic progenitor cells (HPCs) were obtained by means of an iPS bag from iPS cells (SeV2: obtained by introducing c-MYC, OCT3 / 4, SOX2 and KLF4 into human neonatal fibroblasts using the Sendai viral vector according to the method described in WO 2010 / 134526) in a semi-confluent state and maintained in a 6 cm dish, in which MEF was dispersed at a concentration of 3 × 10 ⁵ cells / dish. More specifically, iPS cells were separated using human trypsin solution, and approximately 1/30 to 1/50 cells were plated on C3H10T1 / 2 (available from Riken, Japan) treated with mitomycin C (MMC) in the form of a colony mass. In addition, MMC-treated C3H10T1 / 2 were prepared by plating on a 10 cm dish at a concentration of 8 × 10 ⁵ cells / plate per day the day before iPS cell plating. After seeding, culture was started in Eagle's Minimum Medium (EBM) containing 20 ng / ml VEGF under 5% O2 and 5% CO2 at 37 ° C (day 0). Wednesday was replaced with the same medium on day 3 and day 6.

[0074] On day 7, the culture was continued in an atmosphere of 20% O2 and 5% CO2 at 37 ° C. Wednesday was replaced with the same medium on day 9, day 11 and day 13. On day 14, cells were physically harvested using a cell scraper or pipette tip, and uniformly sized cells were harvested by passing through a 40 micrometer cell sieve. The harvested cells were confirmed as hematopoietic progenitor cells (HPC) based on cell size.

[0075] On day 14, the harvested HPCs were plated into MMC-treated C3H10T1 / 2 at a concentration of 3 x 10 ⁴ to 1 x 10 ⁵ cells / well. The medium used was EBM containing SCF at a concentration of 50 ng / ml, TPO at a concentration of 50 ng / ml and doxycycline at a concentration of 0.5 μg / ml. Next, c-MYC and BMI1 were introduced into the HPC using a lentiviral vector. The lentiviral vector used was an inducible vector controlled by tetracycline and was obtained by recombining the mOKS cassette LV-TRE-mOKS-Ubc-tTA-I2G with c-MYC or BMI1 (LV-TRE-c-MYC-xL-Ubc-tTA-I2G or LV-TRE-BMI1-Ubc-tTA-I2G, respectively) (Nakamura S, et al, Cell Stem Cell. 14: 535-548, 2014). Viral particles used for infection were obtained by infecting 293T cells using a lentiviral vector (MOI 300). Protamine was added only during infection. Next, the medium was changed every other day, and C3H10T1 / 2 and the medium were changed once or twice a week.

[0076] BCL-xl was administered at an MOI of 10 using a lentiviral vector two weeks after administration of c-MYC and BMI1. The lentiviral vector used to administer BCL-xl was an inducible vector controlled by tetracycline and was obtained by recombination of the mOKS cassette to contain BCL-xl in the same manner as described above (LV-TRE-BCL-xL-Ubc-tTA- I2G) (Nakamura S, et al, Cell Stem Cell 14: 535-548, 2014). Protamine was added only during infection. Further, cultivation was performed in EBM containing SCF at a concentration of 50 ng / ml, TPO at a concentration of 50 ng / ml and doxycycline at a concentration of 0.5 μg / ml in 10T1 / 2 feeder cells on a 10 cm dish to obtain megakaryocyte progenitor cells (also referred to as imMKCL).

Reference example 1

[0077] Limiting dilution method

Megakaryocyte progenitor cells (imMKCL) were seeded into a 96-well plate at a density of 1.5 cells / 300 μl / well, followed by culturing for 10 days to 14 days in Iscove's Modified Dulbecco's Medium (IMDM) containing 15% fetal bovine serum (FBS), human SCF (R&D Systems) at 50 ng / ml, TPO at 50 ng / ml, doxycycline (Clontech) at 5 mg / ml and puromycin (Sigma-Aldrich) at 2 mg / ml in an atmosphere of 5% CO2 at 37 ° C. The culture was continued in the same manner after transferring the contents of each well to a 24-well plate and a 6-well plate in order to scale up the culture. The cells in each well were designated as clones of megakaryocyte progenitor cells.

[0078] Analysis of clones of megakaryocyte progenitor cells (first round)

Each of the megakaryocyte progenitor cell clones obtained according to the method described above were washed twice using PBS, plated into a 6-well plate at a concentration of 4 × 10 ⁵ cells / 3 ml, and cultured in an IMDM containing human SCF at a concentration of 50 ng / ml, human TPO at 50 ng / ml, SR1 (Calbiochem) at 750 nM and 15% FBS. The supernatant was collected 7 days later, followed by assessment of the number of formed platelets and platelet function. The estimation of the number of formed platelets was carried out as described below. Namely, antibodies bound to a fluorescent dye, CD41 (BioLegend), CD42a (eBioscience) and CD42b (BioLegend) and propidium iodide (Sigma-Aldrich) were added to the culture supernatant and incubated for 30 minutes, followed by FACSVerseO analysis (B Biosciences). Assay, including exclusion of megakaryocyte progenitor cells based on size, followed by counting cells positive for CD41, CD42a and CD42b and counting platelets per megakaryocyte precursor cell. Platelet function was assessed as described below. Namely, antibodies bound to the fluorescent dye, CD41, CD42b and activated glycoprotein (GP) IIb / IIIa (PAC-1; BD Biosciences) and 0.4 mM phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich) was added to the culture supernatant and incubated for 30 minutes, followed by FACSVerseO analysis. In the assay, the expression level of the activated GP IIb / IIIa was used for evaluation by measurement as fluorescence intensity (MFI).

[0079] As a result of assessing the platelet production volume and platelet function for 22 clones of megakaryocyte progenitor cells in accordance with the method described above, it was confirmed that the platelet production volume was 1.6 times to 1.9 times higher than the control (cells pre-clone megakaryocytes) for clone 1, clone 4 and clone 13 (FIG. 1A). With regard to platelet function, the highest level of activity was demonstrated by clone 13 and was shown to respond well to PMA stimulation compared to control (Fig. 1B).

[0080] Analysis of clones of megakaryocyte progenitor cells (second round)

Five clones (1, 2, 4, 11 and 13) that showed high platelet production and platelet function in the first round results were re-analyzed in the same manner. For clone 4 alone, it was confirmed that the volume of platelet formation was approximately 1.3 times higher than the control (Fig. 2A). In addition, although it was confirmed that platelet function was approximately 3.7 times higher than control for clone 13, the results on platelet function from clones 1, 2, 4 and 11 were different from the results of the first round of the assay, which showed no change. compared to control (Fig. 2B).

[0081] Analysis of clones of megakaryocyte progenitor cells (third round)

The analyzes were performed again in the same manner as for the results of the second round of analysis. There was no change in platelet production versus control (FIG. 3A). In addition, although it was confirmed that platelet function was 1.7 times and 1.6 times higher, respectively, than in the control for clones 4 and 13, the differences were small (Fig. 3B).

[0082] In accordance with these results, it was confirmed that clones of megakaryocyte progenitor cells obtained by the limiting dilution method did not show stable functioning in terms of platelet-forming ability and platelet-generated. Thus, it has been suggested that the limiting dilution method is unsuitable for cloning megakaryocyte progenitor cells.

Example 1

[0083] Cloning megakaryocyte progenitor cells by reprogramming

iPS cells (secondary iPS cells) were obtained by reprogramming megakaryocyte progenitor cells obtained using the previously described method, followed by cloning using secondary iPS cells and again inducing cell differentiation into megakaryocyte progenitor cells to clone these cells (secondary megakaryocyte progenitor cells ) (Fig.4A). Below is a detailed description of it.

[0084] After the introduction of four types of episomal plasmid vectors (pCXLE-hOCT3 / 4-shp53-F, pCXLE-hSK, pCXLE-hUL and pCXWB-EBNA1; Okita K, et al., Stem Cells. 31: 458-66, 2013 and Okita K, et al., Nat Methods. 8: 409-12, 2011) into 1x106 cells of a primary cultured megakaryocyte progenitor cell line by electroporation using an Amaxa Nucleofector, cells were plated at a concentration of 3x105 cells / plate per feeder cells in the form of MEF at a concentration of 1-2 x 105 cells / 6 cm dish. The resulting colonies were removed from the dish 14 days later and subjected to expansion culture, after which 10 secondary clones of iPS cells were used in the assay.

[0085] Analysis of secondary clones of iPS cells

Genomic DNA was extracted from 10 secondary clones of iPS cells obtained according to the method described above, and quantitative PCR was performed on c-MYC and BMI1 within exons using primers compatible with the PCR reaction. As a result, checking the number of insertions of exogenous c-MYC and BMI1 other than two characteristic copies revealed that the number of insertions of exogenous BMI1 in 10 types of secondary clones of iPS cells ranged from approximately 7 copies to 26 copies, while the number of insertions of exogenous c- MYC was approximately 1 copy to 6 copies, and all were different (Fig. 4B).

[0086] Genomic DNA was extracted from 10 secondary clones of iPS cells (using some clones that were different from previously described) obtained in accordance with the method described above, followed by analysis of the entire genomic sequence, and when the number of insertions of exogenous c-MYC and BMI1 was verified, it was confirmed that they inserted into chromosomes in the ratios shown in the following Table 1. It was confirmed that the secondary clones of iPS cells contain approximately 3 times to 5 times the number of BMI1 inserts compared to the number of c-MYC inserts.

[Table 1]

BMI / MYC 2nd-iPS_1 3.90 2nd-iPS_4 2.89 2nd-iPS_11 4.97 2nd-iPS_13 3.47 2nd-iPS_14 3.97 2nd-iPS_15 2.95 2nd-iPS_16 4.75 2nd-iPS_19 2.94 2nd-iPS_20 4.27 2nd-iPS_22 4.89

[0087] According to the results presented above, although the number of exogenous gene insertions introduced at the stage of obtaining megakaryocyte progenitor cells was different for each megakaryocyte progenitor cell, if they were inserted at a constant ratio, it was assumed that progenitor cells were formed megakaryocytes.

[0088] Induction of secondary clones of megakaryocyte progenitor cells from secondary clones of iPS cells

HPC clones were respectively obtained by means of an iPS bag 14 days after three secondary clones of iPS cells (# 4, # 11 and # 12) according to the previously described method. The resulting HPC clones were plated into 6-well plates at a concentration of 1 × 10 ⁵ cells / well, followed by culturing for 27 days in EBM containing SCF at a concentration of 50 ng / ml, TPO at a concentration of 50 ng / ml and doxycycline at a concentration of 0, 5 μg / ml to obtain secondary clones of megakaryocyte progenitor cells. Analysis of the three megakaryocyte progenitor cell clones obtained revealed that the clones were positive for CD41a and CD41b, negative for CD235, and were obtained in the form of a uniform cell group (Fig. 5A and 5B).

[0089] Obtaining HLA-deficient (HLA-null) secondary clones of iPS cells

The target vector shown in FIG. 6A was introduced into a secondary clone of iPS cells (# 11), prepared according to the method described above to remove β2-Microglobulin exon 1 (β2m) by homologous recombination. Whether homologous recombination was performed properly or not was determined by confirming the PCR products using primers designed at the sites indicated in FIG. 6A for wild type and mutant (FIG. 6B). As a result, β2m-deficient secondary iPS cell clones were identified for two clones (clones 3 and 4).

[0090] Further, β2m-deficient secondary clones of megakaryocyte progenitor cells were induced in accordance with the previously described method. In addition, platelets were obtained from the supernatant according to the previously described method. When flow cytometry was performed on β2m-deficient secondary clones of megakaryocyte and platelet progenitor cells using anti-β2m (BD Pharmingen) and HLA (BD Pharmingen) antibodies, all were confirmed to be β2m and HLA deficient (FIG. 7A).

[0091] Confirmation of platelet function of β2m-deficient secondary clones of megakaryocyte progenitor cells

After stimulation of platelets present in culture, the supernatant of the secondary clone of iPS cells of wild type (# 11) and β2m-deficient secondary clones of megakaryocyte progenitor cells with PMA in the same way as described previously, the clones were contacted with the CD42a antibody, CD42b antibody and PAC1. and when positive levels of CD42a, CD42b and PAC1 were measured and assessed on the basis of fluorescence intensity (MFI), there were no observed differences between the wild type and β2m-deficient type (Fig. 7B). Based on the above, it was shown that β2m-deficient platelets have the same function as wild-type platelets.

Example 2

[0092] Search for iPS cell marker suitable for induction of megakaryocyte progenitor cells

mRNA was extracted from two types of secondary iPS cells (referred to as good-iPSC) capable of forming imMKCL prepared according to the previously described method and from two types of iPS cells (referred to as bad-iPSC) unable to form imMKCL, obtained according to by the previously described method, followed by averaging and analysis of the expression levels of the two types of good-iPSC and bad-iPSC using a microchip using Illumina HumanHT-12 v4.0 or Affymetrix Gene Chip Human Gene 2.0 ST Array according to the guidelines provided by the manufacturers (Fig. 8A and 8B).

[0093] In addition, hematopoietic progenitor cells (HPCs) were induced from good-iPSC and bad-iPSC using the same method as previously described (respectively referred to as good-HPC and bad-HPC) followed by analysis using microchip using Illumina HumanHT-12 v4.0 or Affymetrix Gene Chip Human Gene 2.0 ST Array (Figs. 8A and 8B).

[0094] As a result of analysis using the above microarrays, it was confirmed that MEG3to is highly expressed by both good-iPSC and good-HPC. Thus, in the secondary clones of iPS cells and HPCs induced therefrom, it was assumed that confirmation of MEG3 expression would provide for the selection of secondary clones of iPS cells suitable for the induction of megakaryocyte progenitor cells.

Example 3

[0095] Proliferative capacity of megakaryocyte progenitor cells derived from secondary clones of iPS cells

The cellular proliferative capacity of megakaryocyte progenitor cells obtained from the secondary clone of iPS cells was compared with the cell proliferative capacity of the megakaryocyte progenitor cell line before cloning to test the cloning effect of the present invention. In this experiment, a clone derived from 2nd-iPS_11 listed in Table 1 was used for a secondary clone of iPS cells. More specifically, an HPC clone obtained from 2nd-iPS_11 by means of an iPS bag after 14 days according to the previously described method was plated into a 6-well dish at a concentration of 1 × 10 ⁵ cells / well, followed by culturing for 27 days in EBM. containing SCF at a concentration of 50 ng / ml, TPO at a concentration of 50 ng / ml and doxycycline at a concentration of 0.5 μg / ml, to obtain a secondary clone of megakaryocyte progenitor cells (clone 2). Pre-cloning megakaryocyte progenitor cells (parental) obtained in the section entitled "Preparation of megakaryocyte progenitor cells" were used as a comparative example. These cells were plated into a 6-well dish at a concentration of 1 × 10 ⁵ cells / well and cultured for 15 days in EBM containing SCF at 50 ng / ml, TPO at 50 ng / ml and doxycycline at 0.5 μg. / ml.

[0096] As a result of periodic cell counts during the 15 day culture period, megakaryocyte progenitor cells derived from secondary clones of iPS cells clearly showed faster growth than megakaryocyte progenitor cells prior to cloning. The results are shown in FIG. 9A and 9B.

[0097] Maturation capacity of megakaryocyte progenitor cells derived from a secondary clone of iPS cells

A secondary clone of megakaryocyte progenitor cells (clone 2) cultured for 15 days was cultured under conditions to promote the maturation of megakaryocytes (including the addition of SCF at a concentration of 50 ng / ml and TPO at a concentration of 50 ng / ml to EBM followed by the addition of SR-1 ( StemRegenin1) (Selleckchem) at 750 nM and Y27632 (Wako) at 10 μM). The megakaryocyte progenitor cells (parental) were also allowed to mature to megakaryocytes under the same culture conditions as the secondary megakaryocyte progenitor cell clone. Images of both cells photographed by time-lapse photography are shown in FIG. 10A, while the results of image analysis using ImageJ are shown in FIG. 10B. Based on these results, megakaryocytes cloned in accordance with the present invention were presented to demonstrate a higher maturation ability (cellular hypertrophy) than megakaryocytes obtained from original cell clones.

[0098] The ability of megakaryocyte progenitor cells derived from a secondary clone of iPS cells to form platelets

The number of cells that formed platelets was measured visually in the images shown in FIG. 10A to compare the platelet-forming ability from obtained megakaryocytes. As a result, megakaryocytes obtained from cells cloned in accordance with the present invention clearly demonstrated a higher platelet-forming ability (number of prothrombocytes formed) (**: p <0.01).

Claims (19)

1. A method for obtaining clones of secondary stem cells from precursor cells of megakaryocytes, which contains the steps: (i) dedifferentiating megakaryocyte progenitor cells to form a stem cell colony, where megakaryocyte progenitor cells differentiate from stem cells that express at least one exogenous gene selected from the group consisting of oncogenes, polycombus genes, and apoptosis suppressor genes; and (ii) isolating secondary stem cells carrying at least one exogenous gene inserted into their chromosome from the stem cell colony formed in step (i). 2. The method of claim 1, further comprising comparing the efficiency of inducing differentiation of isolated clones of secondary stem cells into megakaryocyte progenitor cells with the efficiency of inducing differentiation of stem cells before cloning. 3. The method according to claim 1, wherein the oncogenes are genes of the MYC family, the polycombus genes are Bmi1 and the apoptosis suppressing genes are BCL-XL genes. 4. The method according to claim 1, wherein the secondary stem cells expressing MEG3 are isolated. 5. The method according to claim 1, wherein the exogenous gene is operably linked to a drug responsive promoter. 6. The method according to claim 1, wherein the dedifferentiation in step (i) is carried out by introducing a reprogramming factor selected from the group consisting of OCT3 / 4, SOX2 and KLF4. 7. A method of obtaining precursor cells of megakaryocytes, which contains the stage: (i) dedifferentiating megakaryocyte progenitor cells to form a stem cell colony, where megakaryocyte progenitor cells differentiate from stem cells that express at least one exogenous gene selected from the group consisting of oncogenes, polycombus genes, and apoptosis suppressor genes; and (ii) isolating secondary stem cells carrying at least one exogenous gene inserted into their chromosome from the stem cell colony formed in step (i); (iii) induction of differentiation of the isolated clones of secondary stem cells into megakaryocyte progenitor cells. 8. A method for obtaining platelets, which contains the steps: (i) dedifferentiating megakaryocyte progenitor cells to form a stem cell colony, where megakaryocyte progenitor cells differentiate from stem cells that express at least one exogenous gene selected from the group consisting of oncogenes, polycombus genes, and apoptosis suppressor genes; (ii) isolating secondary stem cells carrying at least one exogenous gene inserted into their chromosome from the stem cell colony formed in step (i); (iii) induction of differentiation of the isolated clones of secondary stem cells into megakaryocyte progenitor cells; and (iv) ensuring the maturation of the differentiation-induced megakaryocyte progenitor cells into megakaryocytes and the release of platelets. 9. The method of claim 8, further comprising initiating stem cell clones to be HLA deficient. 10. The method of claim 8, wherein the stem cells that express at least one exogenous gene are hematopoietic progenitor cells.

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